Various methods of treating diseases attributed to an inherited or an acquired genetic defect, namely a genetic disease, have been developed. Gene therapy is one such method for treating a genetic disorder fundamentally by replacing a defective gene with a normal gene or complementing a normal gene.
At present, the most clinically and fundamentally developed method of introducing a gene comprises linking a short DNA fragment like cDNA to a downstream site of an ectopic enhancer or promoter that originally does not exist upstream of the gene, introducing the resulting DNA fragment into a cell using a virus or liposome, and allowing the cell to express the gene. This method is easy to manipulate because a short DNA fragment is used. Furthermore, it has a relatively high success rate of introducing a gene into a cell. However, there are some disadvantages. First, it is difficult to control the expression of an introduced gene. In this method, the expression control of a desired gene in a human in vivo is difficult because the promoter and enhancer used are derived from viruses. Second, the existing pattern of an introduced gene in a cell is not stable. Untargeted genes may be destroyed or an excessive number of introduced genes may exist. This is because the introduced gene in a cell may be randomly integrated into a chromosomal DNA. Alternatively, the introduced gene may independently exist extrachromosomally and be maintained without being controlled by DNA synthesis during the S phase or chromosome separation during the M phase of a cell. This fact makes it difficult to control expression of a therapeutic gene in gene therapy and to exhibit therapeutic effects continuously.
For example, sickle cell anemia and thalassemia drew the most attention in the early 1980s as targets for gene therapy. In spite of numerous patients with these diseases, it is not being studied much at present because it is difficult to strictly control the expression of the therapeutic gene (a globin gene) to be introduced. Furthermore, since processing of a huge DNA molecule like a globin gene using restriction enzymes is limited, homologous recombination using yeast is more effective than recombination using an E. coli plasmid. A stable chromosome can thus be prepared in a yeast cell. If such a chromosome is capable of replicating in a human cell, it can be used for the most ideal gene therapy. In the field of gene therapy, it is essential to develop a vector system in which a human gene with an expression control region can function and be stably maintained in a human cell.
An “artificial chromosome” that is a yeast artificial chromosome (YAC) vector has been developed. A long-chain DNA molecule such as a gene ligated to a promoter region and/or an enhancer region can be introduced into this vector and stably maintained following the mechanisms of DNA replication and separation in yeast. A DNA fragment requires three functional structures, a centromere, a DNA replication origin, and a telomere, to function as a chromosome. Based on this fact, the YAC vector has been constructed to contain genes for these three functional structures.
However, the above yeast functional structures do not function in mammals, including humans. Therefore, the functional structures from a mammal or those modified to a mammalian type structure must be used to constitute an artificial chromosome that functions in a mammalian cell.
A centromere, a DNA replication origin, and a telomere of yeast each consist of a several kb DNA sequence whose functions have been well analyzed. In contrast, a centromere of a mammal, especially of a human, is a huge DNA molecule in which a repetitive sequence called the alphoid sequence repeats over several hundred kb or more. In addition, even the primary structure of a mammalian DNA replication origin has not been clarified. Thus, the analysis of functional structures in a mammal is far behind that in yeast, and an artificial mammalian chromosome has yet to be constructed.
Mammals, including humans, commonly have a 5′-TTAGGG-3′ sequence as a repetitive unit of a telomere sequence.